Environmental Pollution 146 (2007) 120e127www.elsevier.com/locate/envpol

Phytoextraction of excess soil phosphorus

Nilesh C. Sharma, Daniel L. Starnes, Shivendra V. Sahi*

Department of Biology, Western Kentucky University, 1906 College Heights Boulevard #11080, Bowling Green, KY 42101-1080, USA

Received 27 October 2005; received in revised form 11 May 2006; accepted 15 June 2006

Crop plants such as cucumber, squash and sunflower accumulate phosphorus and thuscan be used in the phytoextraction of excess phosphorus from soils.

Abstract

In the search for a suitable plant to be used in P phytoremediation, several species belonging to legume, vegetable and herb crops were grownin P-enriched soils, and screened for P accumulation potentials. A large variation in P concentrations of different plant species was observed.Some vegetable species such as cucumber (Cucumis sativus) and yellow squash (Cucurbita pepo var. melopepo) were identified as potential Paccumulators with >1% (dry weight) P in their shoots. These plants also displayed a satisfactory biomass accumulation while growing on a highconcentration of soil P. The elevated activities of phosphomonoesterase and phytase were observed when plants were grown in P-enriched soils,this possibly contributing to high P acquisition in these species. Sunflower plants also demonstrated an increased shoot P accumulation. Thisstudy shows that the phytoextraction of phosphorus can be effective using appropriate plant species.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Phytoextraction; Phosphorus; Crop plants; Phytase; Phosphomonoesterase

1. Introduction

Phosphorus (P) content of soil presents a differing scenario indifferent regions of the world. While many tropical regions withlow-input systems of agriculture are faced with low availability ofsoluble P, some temperate regions with intensive animal-basedagriculture have to deal with excessive P in the soil that is threat-ening the ecosystem. In many parts of the United States and Eu-rope, where enormous quantities of nutrient-rich manures(chicken, swine litter, and other animal wastes) are spread overthe soils, P in manures often exceeds crop requirements (Tarkal-son and Mikkelsen, 2003; Koopmans et al., 2004). The excess ma-nure phosphorus leads to rising soil test P concentrations and anaccelerated loss of P to surface waters (Sims et al., 2000). Soilscontaining >45 mg P/Kg (measured as P2O5) are described asexcessive in P (Delorme et al., 2000). Water-soluble phosphorus

* Corresponding author. Tel.: þ1 270 745 6012; fax: þ1 290 745 6856.

E-mail address: shiv.sahi@wku.edu (S.V. Sahi).

0269-7491/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envpol.2006.06.006

that comes from runoff is the prime cause of eutrophication of theaquatic environment, which is a growing environmental problemworldwide (Sharpley et al., 2001). Besides the long-standingproblem of eutrophication, the transport of P from agriculturalfields has been cited as a factor contributing to the emergenceof a dinoflagellate, Pfiesteria spp. in waterways, which has beenlinked to fish-kills and human health problems (Burkholder andGlasgow, 1997). This environmental issue may dictate the futureexpansion of animal-based agricultural practices and necessitatesfinding ways to reduce nonpoint-source phosphorus pollution.

Various methods are being attempted to reduce soil P andhalt the loss of P in runoff. Application of chemical amend-ments, such as lime, ferric chloride, or alum to soils enrichedwith P is one of such methods used recently (Moore andMiller, 1994; Codling et al., 2002; Dou et al., 2003). Chemicalamendments do not prevent the accumulation of P in soils butmerely reduce the amount of water-soluble phosphorus, thusregulating the runoff loss (Hunger et al., 2005). Moreover, Pimmobilization in soil by these amendments may not be stableon a long-term basis and, instead result in higher soluble

121N.C. Sharma et al. / Environmental Pollution 146 (2007) 120e127

phosphates as in case of Ca and ferric phosphate-dissolutionunder certain normal soil conditions (Smith et al., 2001). An-other strategy to address the excess manure P involves thetreatment of animal feed with additives such as phytase andvitamin D that can increase the digestibility of P in diet (Councilfor Agricultural Science and Technology, 2002). However,concerns have been raised that although phytase can decreasetotal P in litter, it could increase the water-soluble phospho-rus in the litter and hence the potential for P losses to surfacewaters following land application (Vadas et al., 2004).

Alternatively, phytoextraction, plant-assisted removal ofwater-soluble P, could be an attractive strategy. Mining ofsoil P, which includes harvesting P taken up from the soil bya crop grown without external P application, has been proposedas a possible management strategy for P-enriched soils(Frossard et al., 2000; Novak and Chan, 2002). Plant-basedclean-up strategy offers a number of advantages overtraditional clean-up methods and there are several reports ofphytoremediation of heavy metal and organic pollutants(Cunningham et al., 1997; Cao et al., 2003; Kim et al., 2003).However, the ability of vegetation to assist in the remediationof P remains largely unknown. Current P uptake rates are lowfor common row crops and forage grasses used to remove Pfrom soil (Whitehead, 2000). It is felt that the present croppingsystems will require several decades, at the normal rate of P re-moval by plants, to reduce high P concentrations to an environ-mentally safe level. Thus it is important to develop a method forthe rapid removal of soil phosphorus. It has also been suggestedthat for the success of P mining, the phytoremediation strategyshould include plants that can accumulate P manifold higher(>1% dry weight [DW]) than the P content of common plants(Novak and Chan, 2002). Studies reflect the usefulness of plant-assisted P remediation, but no phosphorus hyperaccumulatorhas been identified (Koopmans et al., 2004; Pant et al., 2004).Currently, pasture crops such as Marshall and Gulf ryegrasswere shown to accumulate P (�1% DW) under optimal condi-tions of media and soil (Sharma et al., 2004; Sharma and Sahi,2005). In an attempt to identifying a P hyperaccumulator,several plants belonging to legume, vegetable and herb(foliage) crops were screened in P-enriched soils in the green-house. Our goal was to identify economically valuable cropspecies that contain both high biomass and high shoot P. Thepromising plant species were also characterized for P accumu-lations under different soil P concentrations.

Plants have developed a range of mechanisms to increasethe availability of soil P. These include the development ofhighly branched root systems, release of root exudates, andsecretion of root phosphatases (Schachtman et al., 1998;Raghothama, 1999). Production of phosphatases is a potentiallyimportant way for plants to enhance P availability, as a largeproportion of soil P occurs in organic forms (Richardsonet al., 2004). Phosphatases are required for the mineralizationof organic forms of soil P to release phosphate- the form of Preadily acquired by plants. To understand the role of phospha-tases in P acquisition by the potential P accumulators, the activ-ities of total acid phosphomonoesterase and 6-phytase werealso investigated.

2. Materials and methods

2.1. Seed germination

Seeds of Kentucky wonder bean, blue lake bean, tender green, royal burgundy,

bush wax, pinto bean, kidney bean (Phaseolus vulgaris cultivars), lima bean (Pha-

seolus lunatus), black-eyed bean (Vigna unguiculata subsp. unguiculata), chick-

pea (Cicer arietinum), moong bean (Vigna radiata), lentil (Lens esculenta), pea

(Pisum sativum), soybean (Glycin max), cauliflower (Brassica oleracea var. botry-

tis), cabbage (Brassica oleracea var. capitata), broccoli (Brassica oleracea var.

italica), tomato (Lycopersicon esculentum), egg plant (Solanum melongena), bitter

gourd (Momordica charantia), sponge gourd (Luffa cylindrica), star luffa (Luffa

aegyptica), slicing cucumber (Cucumis sativus), yellow squash (Cucurbita pepo

var. melopepo), edible morning glory (Ipomoea aquatica), carrot (Daucus carota),

radish (Raphanus sativus), okra (Abelmoschus esculentus), mammoth sunflower

(Helianthus annus), spinach (Spinacea oleracea), lettuce (Lactuca sativa), parsley

(Petroselinum crispum), English thyme (Thymus vulgaris), cilantro (Coriandrum

sativum), dill (Anethum graveolens), basil (Ocimum basilicum), pigweed (Amar-anthus spp.) and goosefoot (Chenopodium spp.) were procured from different

sources. The plant species and types were selected on the basis of literature show-

ing a suitable adaptation of some of these crops to a phosphatic clay soil rich in P

(Stricker, 2000). Seeds were sterilized with sodium hypochlorite (1% v/v) and

rinsed several times with sterile deionized water. Seeds were germinated in

a seed bed containing sterilized Jiffy mix and maintained at 25� 2 �C under

12:12-h light/dark regime in a growth chamber. Two-week-old seedlings were

used for transplantation in pot soils.

2.2. Growth of seedlings in P-enriched soil

The pot experiment was carried out in the greenhouse using pots filled with

2 kg of sterilized soil. To simulate P-impacted soils, pot soils were enriched

with an application of 0e2.5 g KH2PO4 kg�1 soil, 8 weeks before transplanta-

tion of seedlings. Soils were also mixed with sand (4 parts soil and 1 part sand)

to reduce the compaction. The soil sample used in this study belonged to the

Pembroke series, and had characteristics of Mollic epipedon (Sharma and

Sahi, 2005). Addition of 0e2.5 g KH2PO4 kg�1 soil results in the extraction

of 4.9e24.3 mg water-soluble P kg�1 soil (Sharma and Sahi, 2005). Seedlings

were transplanted in P-enriched soils at the rate of one seedling pot�1. Each

treatment was replicated three times and repeated twice unless indicated other-

wise. Pots were randomized in a complete block design. Plants were kept in

a greenhouse with 16 h of sunlight, and they were watered four times

a week or as required. The temperature varied from 18 to 20 �C at night

and from 22 to 25 �C during the day unless otherwise indicated. Pot plants

were fertilized with modified half strength Hoagland mixture (Sharma et al.,

2004) every week, and harvested after 8 or 12 weeks. For measurement of bio-

mass growth, harvested plant parts (aerial parts 2 cm above the ground) were

dried for 1 h in air before the fresh weight was measured (g plant�1).

2.3. Analysis of P in plant tissue

Plants from different treatments were harvested after 8 or 12 weeks, and

washed thoroughly with deionized water, divided into root and shoot biomass,

and dried in an oven at 70 �C for 2 days. The ground samples were then

weighed and placed in 15-ml Teflon beakers. Three milliliters of concentrated

HNO3 was added to the sample and the beaker was placed on a hotplate set at

100 �C overnight, until evaporated to dryness. The samples were allowed to

cool and made up gravimetrically to a volume of 20 ml with 2% HNO3. A

VG Elemental Plasma Quad (model PQZ) ICP-AES was used for all data

acquisition. Analyses were performed using an external calibration procedure

and internal standards were included to correct for matrix effects and instru-

mental drift corrections (Schneegurt et al., 2001).

2.4. Phosphomonoesterase assay

Plants harvested after 8 weeks of growth in soils containing either 0 or

2.5 g KH2PO4 kg�1 were washed thoroughly with DI water followed by rinse

122 N.C. Sharma et al. / Environmental Pollution 146 (2007) 120e127

in 2-morpholinoethanesulfonic acid, monohydrate (MES) buffer solution (pH

5.5). Roots were separated, chilled on ice, and homogenized with a mortar and

pestle in 15 mM MES buffer (pH 5.5, 0.5 mM CaCl2 $ H2O, 1 mM EDTA).

The buffer was added at a ratio of 1:5 (root fresh weight/extraction buffer

volume). The extract was centrifuged (13,000 � g; 15 min at 4 �C) and the

supernatant was used for enzyme assay.

For the assay of phosphomonoesterase activity, enzyme extract (50 ml) was

incubated in a total volume of 500 ml of 15 mM MES buffer (pH 5.5, 0.5 mM

CaCl2) in the presence of 10 mM p-nitrophenylphosphate, disodium salt

(SigmaeAldrich, St. Louis, MO) (Richardson et al., 2000). The assay was

conducted over 30 min and reactions were terminated by equal volumes of

0.25 M NaOH. The enzyme activity was calculated from the release of

p-nitrophenol (pNP), determined at 412 nm (relative to standard solutions) by

a UV-vis spectrophotometer (Model Ultrospec 3000, Pharmacia Biotech, USA).

2.5. Phytase assay

Harvested plant roots were treated and homogenized in the above manner.

To assay for phytase activity, 500 ml enzyme extract was incubated in a total

volume of 1 ml 15 mM MES buffer (pH 5.5, 0.5 mM CaCl2) in the presence

of 2 mM myo-inositol hexaphosphoric acid (SigmaeAldrich) (Richardson

et al., 2000). The assay was conducted over 60 min, and reactions were termi-

nated by addition of equal volumes of ice-cold 10% trichloroacetic acid

(TCA). Solutions were subsequently centrifuged to remove precipitated mate-

rial and the phosphate concentration of the solutions were determined by mea-

suring absorbance at 882 nm using the molybdenum-blue reaction (Murphy

and Riley, 1962). Phosphate determinations were recorded at a fixed time

within 1 h following addition of the color reagent to samples, to minimize pos-

sible interference. The enzyme assays were conducted at 26 �C using three

replicates. Phosphomonoesterase and phytase activities were expressed in

mU g�1 root fresh weight (FW), where 1 U is defined as the release of

1 mmol of Pi min�1 under the assay conditions.

2.6. Statistical analysis

The data were analyzed by analysis of variance where F ratios were signif-

icant ( p < 0.05), using SYSTAT (Version 9 for Windows, 1999, Systat

Software Inc., Richmond, CA). Means of plant phosphorus were tested for

significant differences among P treatments.

3. Results

3.1. P accumulation

3.1.1. Bean, vegetable and herb cropsAs shown in Table 1, P concentrations in legume plants var-

ied from 3856 to 8073 mg kg�1 (DW) in root and 3324 to6221 mg kg�1 (DW) in shoot tissues. Only Blue lake beanhad shoot-to-root ratio greater than 1. Among vegetable crops,cole plants accumulated more P in roots (8839 and9154 mg kg�1 DW for cabbage and cauliflower, respectively),while some of the cucurbits had high P in their shoots (up to6730 mg kg1 DW) with shoot-to-root ratio >1 for spongegourd. Cilantro, dill and lettuce, among herbs, had relativelyhigh shoot P, varying from 5433 to 6628 mg kg�1 (DW).

3.1.2. Amaranthus and Chenopodium spp.Table 2 shows accumulation of P in the root and shoot

tissues of Amaranthus and Chenopodium species. Differentgenotypes of Amaranthus cruentus demonstrated high P accu-mulations in shoots, varying from 8521 mg kg�1 (DW) instems to 11,366 mg kg�1 (DW) in leaves. Other species of

Amaranthus had low accumulations of P in their shoots. Che-nopodium quinoa demonstrated accumulations in their leavesgreater (up to 14,839 mg kg�1 DW) than Amaranthus spp.Shoot-to-root P ratio in both Amaranthus and Chenopodiumspp. was greater than 1 or 2 in some.

3.1.3. CucumberCucumber plants grown in soils with increased concentra-

tions of P demonstrated stem accumulations of P greaterthan 13,000 mg kg�1 DW for both periods of time (Fig. 1aand b). Stem accumulations increased up to 18,802 mg kg�1

soil with an increase in soil P, particularly in 8-week plants.However, accumulations in leaves reached 11,002 mg kg�1

DW only after 12 weeks in plants exposed to 2.5 g P kg�1

soil (Fig. 1a and b). Accumulations in fruits also increased(up to 15,373 mg kg�1 DW) with time in both P treatments.Phosphorus contents were high even in control fruits(Fig. 1a and b).

Table 1

Phosphorus content in the shoots and roots of plants evaluated for phytoreme-

diation potential in the greenhouse

Plant Shoot Pa Root Pa Shoot/Root

P ratio

Beans

Kentucky wonder bean 6221 � 549 6927 � 255 0.8

Lima bean 3752 � 299 5156 � 427 0.7

Blue lake bean 4963 � 370 4425 � 421 1.1

Black-eyed pea 3324 � 632 5096 � 296 0.6

Pinto bean 3826 � 264 5343 � 700 0.7

Tender green 3362 � 197 4291 � 222 0.7

Royal burgundy 4361 � 293 8012 � 563 0.5

Bush wax 4145 � 366 8073 � 830 0.5

Chickpea 5111 � 343 6876 � 190 0.7

Moong bean 4992 � 229 4741 � 330 1.0

Kidney bean 4012 � 310 4693 � 531 0.8

Peas 3643 � 167 4597 � 442 0.7

Soybean 3912 � 220 4312 � 435 0.9

Lentil 3990 � 288 3856 � 638 1.0

Vegetable crops

Cauliflower 5291 � 542 9154 � 883 0.5

Cabbage 5088 � 660 8839 � 592 0.5

Broccoli 3316 � 310 3924 � 186 0.8

Tomato 4217 � 368 5946 � 283 0.7

Egg plant 3812 � 739 3712 � 175 1.0

Bitter gourd 4612 � 883 6123 � 326 0.7

Sponge gourd 6730 � 398 5892 � 250 1.1

Star luffa 4625 � 211 8723 � 299 0.5

Edible morning glory 3812 � 339 3612 � 341 1.0

Carrot 4720 � 429 5149 � 333 0.9

Radish 5934 � 200 6789 � 257 0.8

Okra 4612 � 364 6123 � 332 0.7

Herbs

Spinach 6547 � 732 e eLettuce 6628 � 422 8723 � 554 0.7

Parsley 4332 � 328 4418 � 281 0.9

English thyme 3297 � 320 3460 � 334 0.9

Cilantro 5433 � 632 5585 � 280 0.9

Dill 5889 � 392 6486 � 230 0.9

Basil 4322 � 431 3853 � 410 1.1

Plants were grown in soils enriched with 2.5 g P kg�1 soil for 8 weeks.a Values are mg kg�1 dry weight; the mean of three replicates � standard

error of the mean.

123N.C. Sharma et al. / Environmental Pollution 146 (2007) 120e127

3.1.4. SquashLike cucumber, squash had P accumulations varying from

9000 to 15,215 mg kg�1 (DW) in different parts of the shoot(Fig. 2a and b). Accumulations in stem and leaf increasedwith an increase in soil P and period of growth, stem P reach-ing a level of 15,215 mg kg�1 DW after 12 weeks. Increase instem P was conspicuous even in control plants with P over11,000 mg kg�1 DW after 12 weeks. Accumulations in thefruit reached to a level of 14,788 mg P kg�1 DW. High contentof P was also observed in control fruits (Fig. 2a and b).

3.1.5. SunflowerSunflower shoots accumulated with higher contents of P in

leaf and flower (8000e9988 mg kg�1 DW). The pattern ofshoot P accumulation in sunflower was comparable in plantsgrown for 8 weeks and 12 weeks (Fig. 3a and b). The increasein soil P from 1 to 2.5 g P kg�1 soil had no significant( p > 0.05) effect on the accumulation pattern in sunflower.However, roots showed more than 50% higher P contentswhen grown in 2.5 g P kg�1 soil.

3.2. Biomass of cucumber, squash and sunflower

The three plant species grown on a high P concentration(2.5 g kg�1 soil) demonstrated a significant increase( p < 0.05) in biomass over the periods of growth (Table 3).Increase in biomass in these plants was either higher or com-parable to that of control plants.

3.3. Enzyme activities in cucumber, squash andsunflower

Acid phosphomonoesterase activity in cucumber grownin the P-enriched soil was 35% higher than in controls(Table 4). A similar pattern was also recorded in the acidphosphomonoesterase activity of squash grown in P-enriched

Table 2

Accumulation of P in Amaranthus and Chenopodium spp. grown in soils en-

riched with 2.5 g P kg�1 soil for 8 weeks

Plant Stem Pa Leaf Pa Root Pa Shoot/Root

P ratio

Amaranthuscruentus (RRC 1011)

9596 � 583 6737 � 150 7247 � 221 1.1

A. cruentus

(TGR 542)

8521 � 422 11366 � 633 3405 � 124 2.9

A. cruentus(Montana-3)

8904 � 290 9184 � 392 3392 � 226 2.6

A. hybridus

(Pen d 136-1)

6499 � 390 6638 � 290 3909 � 145 1.6

A. tricolor

(RRC 241)

5860 � 167 7352 � 222 4202 � 296 1.5

Chenopodium

quinoa (Chile)

4613 � 560 11565 � 628 3978 � 112 2.0

Chenopodium

quinoa (Chile QQ056)

3272 � 199 14839 � 577 3994 � 210 2.2

a Values are mg kg�1 dry weight; the mean of four replicates � standard er-

ror of the mean.

soils. However, the trend of enzyme activity was different insunflower, the difference in the phosphomonoesterase activitybetween the two groups being statistically insignificant( p > 0.05).

Phytase activity in cucumber grown in the P-enriched soilwas >400% higher than in controls, while in the same groupof squash the activity was higher by >40% (Table 4). How-ever, the phytase activity of sunflower was 26% higher incontrols than in plants grown in P-enriched soils.

4. Discussion

4.1. Plant accumulation of phosphorus

Among the various groups of plants (legumes, vegetablesand herbs), a large variation in P accumulation was noticed,and only few plant spp. demonstrated the P accumulation ata level of 1% (10 g P kg�1 shoot DW) or more in their shoots.

0

5000

10000

15000

20000

25000

Soil P (g/kg soil)

Tissu

e P

(m

g/kg

D

W)

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Control 1 2.5Soil P (g/kg)

Control 1 2.5

Tissu

e P

(m

g/kg

D

W)

Root

Stem

Leaf

Fruit

Root

Stem

Leaf

Fruit

a

b

Fig. 1. (a) Phosphorus accumulation in cucumber shoots and roots grown in

the soil enriched with 0e2.5 g P kg�1 (soil) for 8 weeks. Values are the

mean of 3 replicates � standard error of the mean. (b) Phosphorus accumula-

tion in cucumber shoots and roots grown in the soil enriched with 0e2.5 g

P kg�1 (soil) for 12 weeks. Values are the mean of 3 replicates � standard

error of the mean.

124 N.C. Sharma et al. / Environmental Pollution 146 (2007) 120e127

The plant species and types were selected on the basis of lit-erature showing a suitable adaptation of some of these plantcrops to a phosphatic clay soil that is rich in P (Stricker,2000). Plant species belonging to cole crops and some tropicallegumes were reported to perform exceptionally well in yieldswhen grown in the reclaimed phosphatic clay soil (Stricker,

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

Root

Stem

Leaf

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Tissu

e P

(m

g/kg

D

W)

Tissu

e P

(m

g/kg

D

W)

Control 1 2.5Soil P (g/kg)

Control 1 2.5Soil P (g/kg)

Root a

b

Stem

Leaf

Fruit

Fig. 2. (a) Phosphorus accumulation in squash shoots and roots grown in the

soil enriched with 0e2.5 g P kg�1 (soil) for 8 weeks. Values are the mean

of 3 replicates � standard error of the mean. (b) Phosphorus accumulation

in squash shoots and roots grown in the soil enriched with 0e2.5 g P kg�1

(soil) for 12 weeks. Values are the mean of 3 replicates � standard error of

the mean.

2000), but these plant types demonstrated low P accumulationsin their shoot biomass in this study. These findings are consis-tent with the earlier report (Delorme et al., 2000), whichincluded studies on alfalfa, soybean, red clover, broccoli,collard, corn and other agricultural crops with the highest Pconcentration in collard (6.3 g kg�1 DW) and corn shoots(4.9 g kg�1 DW). However, among herbs, pigweed (Amaran-thus spp.) and goosefoot (Chenopodium spp.) plants were

0

2000

4000

6000

8000

10000

12000

0

2000

4000

6000

8000

10000

12000

14000

Root

Stem

Leaf

Flower

Root

Stem

Leaf

Flower

Tissu

e P

(m

g/kg

D

W)

Tissu

e P

(m

g/kg

D

W)

Soil P (g/kg)

Control 1 2.5

Soil P (g/kg)

Control 1 2.5

a

b

Fig. 3. (a) Phosphorus accumulation in sunflower shoots and roots grown in the

soil enriched with 0e2.5 g P kg�1 (soil) for 8 weeks. Values are the mean of 3

replicates � standard error of the mean. (b) Phosphorus accumulation in sun-

flower shoots and roots grown in the soil enriched with 0e2.5 g P kg�1 (soil)

for 12 weeks. Values are the mean of 3 replicates � standard error of the mean.

Table 3

Fresh weight biomass of cucumber, squash and sunflower plants grown in soils augmented with 0 and 2.5 g P kg�1 soil

Growth period 2.5 g P kg�1 soila Controla

Cucumber Squash Sunflower Cucumber Squash Sunflower

5 weeks 30.6 � 2.60 39.0 � 2.88 30.6 � 2.33 29.6 � 1.66 35.6 � 3.38 26.6 � 0.88

6 weeks 44.0 � 1.00 48.3 � 1.45 50.0 � 1.52 42.6 � 1.76 47.3 � 1.45 52.3 � 1.45

8 weeks 77.3 � 3.84 75.6 � 3.28 72.3 � 1.45 63.6 � 2.02 71.3 � 2.02 75.0 � 5.77

a Values are Fresh weight (g); the mean of three replicates � standard error of the mean.

125N.C. Sharma et al. / Environmental Pollution 146 (2007) 120e127

promising P accumulators, with some of the varieties accumu-lating in the range of 1.1e1.4% P (DW) in their leaves(Table 2). The ratio of shoot-to-root P was also high (2 or>2) in these varieties. On the basis of P contents in aerialparts, these plants can be suitable for P phytoremediation,but some of pigweed and goosefoot spp. are known for theirinvasive nature, and thus can be difficult to remove whennot desired. However, a cautious approach involving harvest-ing of shoots before flowering can be effective in reducingsoil P levels, particularly on fallow lands.

While screening vegetable crops, cucurbits, namely cucum-ber and yellow squash, demonstrated an appreciable P accu-mulation in the aerial parts matching observations on theirtrials in the phosphatic clay soil (Stricker, 2000). With anaccumulation of about 1.8% in stem and 1% in leaves(DW), cucumber can remove a substantial quantity of soil P.Like cucumber, yellow squash also accumulated over 1% (asmuch as 1.4% DW) in their aerial parts. Cucumber stemshowed an increasing accumulation with the increase in soilP concentration after 8 weeks, but leaves had higher accumu-lations depending on soil P after 12 weeks. It is likely thatcucumber stems first acquire P from soil and then distributeit in parts like leaves and fruits after a period of time. Accumu-lation in fruits also increased significantly ( p < 0.05) withtime. The pattern of P accumulation in squash was differentin that stems and leaves both had increasing concentrationsof P with increase in time. However, squash fruits had anaccumulation of about 1.4% (DW), irrespective of P concen-trations in soil. Even control plants grown in soils withoutany external addition of P had high P concentrations in fruits(after 8 weeks) and stems (after 12 weeks). This may be be-cause of high efficiency of squash plants for P acquisitionfrom soils having even low concentrations of P. The concentra-tion of soluble P in soils used for the control was determinedto be <5 mg kg�1 soil (Sharma and Sahi, 2005). It is alsointeresting to note that the biomass growth in these P accumu-lating plants (cucumber and squash) remained unaffected bythe high concentration of P in soil (Table 3) unlike incidencesof toxic symptoms in other plants (Cogliatti and Clakson,1983; Novak and Chan, 2002). It appears that these speciesemploy more efficient P sequestration mechanisms to avoidP toxicity.

Table 4

Acid phosphomonoesterase and 6-phytase activities of root extracts in cucum-

ber, squash and sunflower plants grown in P-enricheda soils for 8 weeks

Treatments Acid phosphomonoesterase

activity

Phytase

activity

Cucumber control (P�) 386 � 59.6b 0.52 � 0.08b

Cucumber (Pþ) 529 � 48.5 2.35 � 0.85

Squash control (P�) 390 � 49.6 1.70 � 0.51

Squash (Pþ) 538 � 53.2 2.41 � 0.63

Sunflower control (P�) 586 � 74.0 3.00 � 0.70

Sunflower (Pþ) 568 � 66.8 2.37 � 0.29

a P was applied at the rate of 2.5 g kg�1 soil.b Values are mU g�1 root fresh weight; the mean of three replicates � stan-

dard error of the mean.

In an earlier study, shoot accumulations of P were deter-mined for oil crops such as Indian mustard, canola and rape oil-seed, and a maximum level of 0.46% P (shoot DW)accumulation was observed in Indian mustard (Delormeet al., 2000). It was interesting to determine the P accumulationpattern in sunflower plants, which is one of the most widelyused edible oil crops world-wide. The result shows that sun-flower accumulates P greater than Indian mustard, canola andrape oilseed with stem, leaf and flower accumulations of 0.6,0.8 and 1.0% (DW), respectively. The effect of soil P concen-tration (>1 g kg�1 soil) and duration of time had no significanteffect on the accumulation potential of sunflower plants.Increase in root P was noticeable with increase in duration oftime. Like cucumber and squash, sunflower also displayedgrowth in biomass in the presence of a high concentration ofsoil P as much as control plants. As this crop is known for itshigh biomass in fields, the cumulative P removal capacitymay be significantly higher at the demonstrated levels of P ac-cumulation in aerial parts. Besides element accumulation andharvestable biomass, what is important to determine the phy-toextraction potential of a plant species is the depth of rootingzone, as suggested by Mertens et al. (2005). Notably, sunflowerwas observed to have extensive growth of root system extend-ing deep into soils (data not presented) under high P conditions.That is also true of Amaranthus and Chenopodium species.

4.2. Activities of phosphomonoesterase and phytase

The long-term use of P fertilizer or animal manure in-creases the P concentration in soil solution and much of theP added in this manner becomes transformed into organicand inorganic forms that are of limited availability to plants(Sanyal and De Datta, 1991). Plants have evolved differentmechanisms to enhance the availability of P, and root leachingof phosphatase enzymes in the soil solution is one of the effec-tive ways. The role of 6-phytaseda phosphomonoesterasewith high specific activity for phytatedin plant P nutritionhas been recently emphasized because phytate constitutes upto 50% of the total organic P in soil (Turner et al., 2002). Inthis backdrop, we analyzed phosphomonoesterase and phytaseactivities in plant species that showed an increased P accumu-lation (cucumber, squash and sunflower). Results in this inves-tigation show the enhanced activities of both enzymes incucumber and squash grown in P-enriched soils as comparedto the activities in controls (with no addition of P). Whenthe activity of phosphomonoesterase increased to about 35%in both (cucumber and squash), the phytase activity increasedabout 400% in cucumber and 40% in squash with respect tocontrols. Sunflower plants, however, demonstrated either com-parable (phosphomonoesterase) or higher activity (phytase)under the P-deficient condition (controls). The pattern ofenzyme activities in cucumber and squash is remarkably dif-ferent to the pattern of activities in sunflower and other plantspecies (Hayes et al., 1999; Richardson et al., 2000). However,this is comparable to the enzyme activity in Marshall and Gulfryegrass that also show higher P accumulations fromP-enriched soils (Sharma and Sahi, 2005). A similar trend

126 N.C. Sharma et al. / Environmental Pollution 146 (2007) 120e127

was also observed in Trifolium repens (a legume pasture),which demonstrated higher phytase activity in high P condi-tions (Hayes et al., 1999). Plants having high phytase activityin their roots can hydrolyze phytates, which account for a largeproportion of unavailable soil P pool, and can thus depleteexcess P source more efficiently. Higher enzyme activities incucumber and squash thus may be one of the factors contrib-uting to their P accumulation efficiency. The molecular mech-anism of P nutrition in plants under the conditions ofP adequacy is not well known; however, much informationis available on the acquisition of P under P deficiency(Raghothama, 1999). Although this study identifies someeconomically important plant species with affinity for highP acquisition, the P accumulation potential of these plantsneeds to be verified under natural conditions of high soil P.

5. Conclusion

A large variation in P accumulation pattern among variouscrop plants was observed in this study. Cucurbits such ascucumber and squash demonstrated high P accumulations(as much as 1.4% DW) in their shoots including fruits. Thepattern of enzyme activities was also interesting in theseplants. When the activity of phosphomonoesterase increasedto about 35% in both, the phytase activity increased about400% in cucumber and 40% in squash with respect to controls.The expression of enhanced enzyme activities in these plantspecies may be one of the factors contributing to their uniqueP acquisition ability under the conditions of high soilP. Sunflower was another crop plant that showed high shootaccumulations, nearly 1% (DW). However, the pattern ofenzyme activities in sunflower was different than in the cucur-bits. Some of the Amaranthus and Chenopodium species,though invasive in nature, were also outstanding accumulatorsof P.

As these P accumulators can generate high biomass andhave economic value, they can be potential candidates for Pphytoextraction in the localized areas of high soil P.

Acknowledgments

This research was carried out with the support from theU.S. Department of Agriculture (Grant 58-6406-1-017). Theauthors duly acknowledge the support and encouragementfrom the Applied Research and Training Program, WesternKentucky University, in carrying out this research.

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